MicroRNAs (miRs) regulate the molecular pathways linked to pain, inflammation, and immune response. Aberrant miR expression is seen in a variety of conditions including acute and chronic pain, inflammation, neurodegeneration, cancer, and others. Acute and chronic pain affects over 100 million US adults, and a large subpopulation of chronic pain patients suffer from >1 pain condition. Recent studies indicate that extracellular miRs (including those in serum-derived exosomes) act as physiologic ligands for rodent toll-like receptor 7 (TLR7; TLR8 in humans). The long-term goal is to better understand the role of extracellular miRs in acute and chronic pain. Pain associated with inflammation of the tooth pulp (pulpitis) is used here as a model of a single acute pain condition. Pain in patients diagnosed with ?3 of the following conditions - temporomandibular disorder (TMD), episodic migraine (EM), vulvar vestibulitis syndrome (VVS), fibromyalgia (FM), and irritable bowel syndrome (IBS) - is used as a model of chronic overlapping pain conditions. The objective of this project is to examine whether serum-derived exosomal miRs contribute to chronic overlapping pain conditions. The central hypothesis is that serum-derived exosomal miRs bind TLR8 and activate NFKB, resulting in the release of pro-pain cytokines. This hypothesis has been formulated on the basis of preliminary data produced in our laboratory. Using pulpitis as a model of acute pain, we show that miRs are differentially expressed in inflamed pulps as compared to normal pulps. Using blood samples of chronic pain patients we show that the miR profile in the blood of patients with chronic pain conditions differs from that o pain-free controls, and that the miR profiles of patients with >1 chronic pain condition differs from those with a single pain condition. Our pilot data also suggest that experimental muscle pain sensitivity and affective pain is correlated with miR expression. These preliminary studies lead us to propose the novel hypothesis that serum-derived exosomal miRs bind TLR8 and activate NFKB, resulting in the release of pro-pain cytokines. Specific Aim 1 examines the miR profile in serum-derived exosomes and affected (local) tissues of three groups of patients (n=150) (50 with symptomatic pulpitis; 50 with asymptomatic pulpitis; and 50 pain-free controls). Specific Aim 2 compares the miR profile in serum-derived exosomes of patients diagnosed with ?3 chronic pain conditions (TMD, EM, VVS, FM, IBS) (n=100) with that of pain-free controls (n=100). Specific Aim 3 explores whether miRs differentially expressed in pain patients bind TLR8 and induce NFKB activation and the release of pro-pain cytokines. This proposal is innovative because it is the first to examine the role of exosomal miRs in overlapping pain conditions. The proposed research is significant because it is the first step in a continuum of research that is expected to lead to the development of miR-based biomarkers and pharmacologic strategies that will result in better diagnosis and management of chronic overlapping pain conditions.